Retroviral RNA encapsidation is a highly ordered process, by which two full-length genomic (g) RNAs are incorporated into assembling virions. Although a single gRNA template can support retroviral reverse transcription and proviral DNA synthesis, recombination and reassortment of polymorphisms is a hallmark feature of the retrovirus and dependent on a diploid genome. Prior to packaging, intergenomic annealing initiates formation of loose noncovalent dimers of unspliced HIV-1 RNA, which is then selected for encapsidation over the excess host cellular and viral spliced HIV-1 RNAs (˜99% of the total cellular RNA). This selectivity is due to the recognition of a cis-acting packaging element within the 5′ untranslated region (5′UTR) by the nucleocapsid (NC) domain of the Gag polyprotein via two zinc fingers. Confocal microscopy studies suggest that the capture of gRNA by Gag occurs in a perinuclear/centrosomal site. A single interaction with gRNA may nucleate Gag multimerization during assembly, or multiple Gag proteins may preassemble into oligomeric arrays in the cytoplasm prior to gRNA binding.
Secondary and tertiary structures of retroviral gRNAs in the 5′UTR as well as proximal coding regions are involved in gRNA dimerization, packaging, and translation. In HIV-1, the canonical packaging signal (also referred to as psi or ψ) contains four stem loops (SL1-SL4) located downstream of the primer binding site (PBS) and extending into the 50 terminus of gag coding sequences. In particular, SL1 contains the dimerization initiation site (DIS) which forms the kissing loop for gRNA dimerization. Both SL2 (containing the splice donor site) and SL3 have high affinity for NC, but only SL3 is recognized as the core packaging element containing the highly conserved GGAG NC-binding sequence. In complex retroviruses such as HIV-1, gRNA packaging and dimerization signals map to multiple sequences in both LTRs and the 5′ end of gag, including the trans-activating-responsive (TAR) stem loop and PBS. Despite attempts to define the complete HIV-1 gRNA packaging signal, the relative role of each sequence within HIV-1 genome has remained debatable.